Larry W. Kraiss

Escaping the Nuclear Confines: Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenecity of human platelets

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

Effect of rosuvastatin therapy on carotid plaque morphology and composition in moderately hypercholesterolemic patients: a high-resolution magnetic resonance imaging trial.

BACKGROUND Magnetic resonance imaging (MRI) can noninvasively assess changes in atherosclerotic plaque morphology and composition. The ORION trial assessed the effects of rosuvastatin on carotid plaque volume and composition. METHODS The randomized, double-blind ORION trial used 1.5-T MRI to image carotid atherosclerotic plaques at baseline and after 24 months of treatment. Forty-three patients with fasting low-density lipoprotein cholesterol > or = 100 and < 250 mg/dL and 16% to 79% carotid stenosis by duplex ultrasound were randomized to receive either a low (5 mg) or high (40/80 mg) dose of rosuvastatin. RESULTS After 24 months, 33 patients had matched serial MRI scans to compare by reviewers blinded to clinical data, dosage, and temporal sequence of scans. Low-density lipoprotein cholesterol was significantly reduced from baseline in both the low- and high-dose groups (38.2% and 59.9%, respectively, both P < .001). At 24 months, there were no significant changes in carotid plaque volume for either dosage group. In all patients with a lipid-rich necrotic core (LRNC) at baseline, the mean proportion of the vessel wall composed of LRNC (%LRNC) decreased by 41.4% (P = .005). CONCLUSIONS In patients with moderate hypercholesterolemia, both low- and high-dose rosuvastatin were effective in reducing low-density lipoprotein cholesterol. Furthermore, rosuvastatin was associated with a reduction in %LRNC, whereas the overall plaque burden remained unchanged over the course of 2 years of treatment. These findings provide evidence that statin therapy may have a beneficial effect on plaque volume and composition, as assessed by noninvasive MRI.

Protein synthesis by platelets: historical and new perspectives

Summary.  In the late 1960s, numerous investigators independently demonstrated that platelets are capable of synthesizing proteins. Studies continued at a steady pace over the next 30 years and into the 21st century. Collectively, these investigations confirmed that platelets synthesize proteins and that the pattern of protein synthesis changes in response to cellular activation. More recent studies have characterized the mechanisms by which platelets synthesize proteins and have shown that protein synthesis alters the phenotype and functions of platelets. Here, we chronologically review our increased understanding of protein synthetic responses in platelets and discuss how the field may evolve over the next decade.

Anucleate platelets generate progeny

Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and alpha-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream.

Outside-In Signals Delivered by Matrix Metalloproteinase-1 Regulate Platelet Function

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix proteins. These enzymes are implicated in a variety of physiological and pathological events characterized by extracellular matrix remodeling. Recent studies suggest that MMPs may have a signaling capacity, but direct evidence supporting this concept is lacking. In the present study, we demonstrate that outside-in signals delivered by exogenous MMP-1 (interstitial collagenase) markedly increase the number of tyrosine-phosphorylated proteins in platelets. Active MMP-1 also targets &bgr;3 integrins to areas of cell contact and primes platelets for aggregation. Examination of the endogenous enzyme demonstrated that activated platelets process latent MMP-1 into its active form. Neutralization of MMP-1 activity with MMP inhibitors or specific blocking antibodies markedly attenuates agonist-induced phosphorylation of intracellular proteins, movement of &bgr;3 integrins to cell contact points, and intercellular aggregation. The finding that MMP-1 is rapidly activated in platelets and controls functional responses identifies a new role for this metalloproteinase as a signaling molecule that regulates thrombotic events.

The Society for Vascular Surgery Vascular Quality Initiative

The Society for Vascular Surgery (SVS) Vascular Quality Initiative (VQI) is designed to improve the quality, safety, effectiveness, and cost of vascular health care. It uses the structure of a Patient Safety Organization to permit collection of patient-identified information but protect benchmarked comparisons from legal discovery. The SVS VQI is uniquely organized as a distributed network of regional quality groups to facilitate local translation of registry data into practice change while maintaining the power of a national registry. Detailed data specific to each commonly performed open and endovascular procedure are collected, both in-hospital and at ≥ 1 year of follow-up. Quality measures are reported to physicians and hospitals, which allow anonymous risk-adjusted benchmarking within regions or nationally. All specialties that perform vascular procedures are included, and international participation is encouraged. This review describes the current status of the SVS VQI.

A Randomized, Controlled Pilot Study of Autologous CD34+ Cell Therapy for Critical Limb Ischemia

Background—Critical limb ischemia portends a risk of major amputation of 25% to 35% within 1 year of diagnosis. Preclinical studies provide evidence that intramuscular injection of autologous CD34+ cells improves limb perfusion and reduces amputation risk. In this randomized, double-blind, placebo-controlled pilot study, we evaluated the safety and efficacy of intramuscular injections of autologous CD34+ cells in subjects with moderate or high-risk critical limb ischemia, who were poor or noncandidates for surgical or percutaneous revascularization (ACT34-CLI). Methods and Results—Twenty-eight critical limb ischemia subjects were randomized and treated: 7 to 1×105 (low-dose) and 9 to 1×106 (high-dose) autologous CD34+ cells/kg; and 12 to placebo (control). Intramuscular injections were distributed into 8 sites within the ischemic lower extremity. At 6 months postinjection, 67% of control subjects experienced a major or minor amputation versus 43% of low-dose and 22% of high-dose cell-treated subjects (P=0.137). This trend continued at 12 months, with 75% of control subjects experiencing any amputation versus 43% of low-dose and 22% of high-dose cell-treated subjects (P=0.058). Amputation incidence was lower in the combined cell-treated groups compared with control group (6 months: P=0.125; 12 months: P=0.054), with the low-dose and high-dose groups individually showing trends toward improved amputation-free survival at 6 months and 12 months. No adverse safety signal was associated with cell administration. Conclusions—This study provides evidence that intramuscular administration of autologous CD34+ cells was safe in this patient population. Favorable trends toward reduced amputation rates in cell-treated versus control subjects were observed. These findings warrant further exploration in later-phase clinical trials. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00616980

National trauma databank analysis of mortality and limb loss in isolated lower extremity vascular trauma.

OBJECTIVES Lower extremity injury is common in trauma patients; however, the influence of arterial injury on devastating patient and limb outcomes can be confounded by the presence and physiological derangement of concomitant head or thoracoabdominal injuries. We analyzed isolated lower extremity injuries with an arterial component. Our aim was to elucidate factors associated with mortality and limb loss in this selected population. METHODS We reviewed trauma incidents from the National Trauma Data Bank (2002-2006) containing isolated lower extremity injury codes and a specified infrainguinal arterial injury. Demographics, injury patterns, clinical characteristics, and adverse outcomes (death, amputation) during initial hospitalization were collected. Multivariate logistic regression was used to identify risk factors for limb loss. RESULTS There were 651 isolated infrainguinal arterial injuries. Death (18) and early limb loss (42) were studied by mechanism (penetrating, n = 431; blunt, n = 220). Half of the deaths involved injury to the common femoral artery (CFA), and over 80% had injury to the CFA or superficial femoral artery (SFA). Death was three times as frequent in the CFA/SFA than in the popliteal/tibial injuries (P = .02). Penetrating injuries were present in almost 80% of deaths, and most of these were gunshot wounds. Patients who died had mean initial systolic blood pressure of 59.7 mm Hg, and almost 40% had no blood pressure on arrival. Mean initial Glasgow Coma Score was 4.5, and almost 80% arrived with a Glasgow Coma Score of 3 despite the absence of head injury. Twenty-seven above- and 15 below-the-knee amputations were performed. The popliteal artery was injured in half of the amputations, with injury isolated to the popliteal or tibial arteries in about three-quarters. Amputation was twice as frequent in popliteal/tibial than CFA/SFA injury (P = .03) and twice as frequent in blunt than penetrating injury (P = .05). Multiple arterial injuries (odds ratio, 5.2; 95% confidence interval, 1.7-15.6; P = .003), and fracture (odds ratio, 2.2; 95% confidence interval, 1.1-4.2; P = .02) independently predicted amputation, while the presence of nerve injury and soft tissue disruption did not. CONCLUSIONS Isolated lower extremity trauma with vascular injury has a nearly 10% rate of mortality or limb loss. Mortality is associated with penetrating mechanism and early shock, likely resulting from prehospital proximal arterial hemorrhage. In contrast, early limb loss is more common with blunt distal vascular injury, especially to the popliteal and tibial arteries. Neither nerve nor soft tissue injury predicted limb loss but may result in delayed amputations not captured in this acute outcomes dataset.

Readmission destination and risk of mortality after major surgery: an observational cohort study

BACKGROUND Hospital readmissions are common after major surgery, although it is unknown whether patients achieve improved outcomes when they are readmitted to, and receive care at, the index hospital where their surgical procedure was done. We examined the association between readmission destination and mortality risk in the USA in Medicare beneficiaries after a range of common operations. METHODS By use of claims data from Medicare beneficiaries in the USA between Jan 1, 2001, and Nov 15, 2011, we assessed patients who needed hospital readmission within 30 days after open abdominal aortic aneurysm repair, infrainguinal arterial bypass, aortobifemoral bypass, coronary artery bypass surgery, oesophagectomy, colectomy, pancreatectomy, cholecystectomy, ventral hernia repair, craniotomy, hip replacement, or knee replacement. We used logistic regression models incorporating inverse probability weighting and instrumental variable analysis to measure associations between readmission destination (index vs non-index hospital) and risk of 90 day mortality for patients who underwent surgery who needed hospital readmission. FINDINGS 9,440,503 patients underwent one of 12 major operations, and the number of patients readmitted or transferred back to the index hospital where their operation was done varied from 186,336 (65·8%) of 283,131 patients who were readmitted after coronary artery bypass grafting, to 142,142 (83·2%) of 170,789 patients who were readmitted after colectomy. Readmission was more likely to be to the index hospital than to a non-index hospital if the readmission was for a surgical complication (189,384 [23%] of 834,070 patients readmitted to index hospital vs 36,792 [13%] of 276,976 patients readmitted non-index hospital, p<0·0001). Readmission to the index hospital was associated with a 26% lower risk of 90 day mortality than was readmission to a non-index hospital, with inverse probability weighting used to control for selection bias (odds ratio [OR] 0·74, 95% CI 0·66-0·83). This effect was significant (p<0·0001) for all procedures in inverse probability-weighted models, and was largest for patients who were readmitted after pancreatectomy (OR 0·56, 95% CI 0·45-0·69) and aortobifemoral bypass (OR 0·69, 95% CI 0·61-0·77). By use of hospital-level variation among regional index hospital readmission rates as an instrument, instrumental variable analysis showed that the patients with the highest probability of returning to the index hospital had 8% lower risk of mortality (OR 0·92 95% CI 0·91-0·94) than did patients who were less likely to be readmitted to the index hospital. INTERPRETATION In the USA, patients who are readmitted to hospital after various major operations consistently achieve improved survival if they return to the hospital where their surgery took place. These findings might have important implications for cost-effectiveness-driven regional centralisation of surgical care. FUNDING None.